VIETNAM NATIONAL UNIVERSITY – HCMC

INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

INTERNSHIP REPORT

Process of collecting ectomycorrhizal root tips

from Pinus kesiya and comparing the hyphal
spreading area of Suillus bovinus’ strains
 Student name: Ton Ngoc Minh Trang

Student ID: BTBTIU18364

Host Institution: The Applied Biotechnology Institute (ABI)

On-site supervisor: Dang Hoang Quyen

IU Advisor: Assoc. Prof. Tran Thi My Hanh

Time: 14.03.2022 – 14.05.2022

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 This report is submitted to:

INTERNATIONAL UNIVERSITY, VIETNAM NATIONAL

UNIVERSITY – HO CHI MINH CITY

Place: The Applied Biotechnology Institute (ABI)

Address: Floor 4, Van Dat Tower, CN8 street, Tan Binh Industrial Park, Tay Thanh Ward, Tan Phu

District, Ho Chi Minh City.

Email: abiotechinst@gmail.com

On-site supervisor: Dang Hoang Quyen

Email: dhquyendl@gmail.com

Phone number: 0984090850

IU Advisor: Assoc. Prof. Tran Thi My Hanh

Email: ttmhanh@hcmiu.edu.vn

Duration of the Internship: 8 weeks (14.03.2022 – 14.05.2022)

3
 ACKNOWLEDGEMENT

Firstly, I desire to show my deepest appreciation for background lectures from all of the instructors at

International University, especially Assoc. Prof. Tran Thi My Hanh gave us many support, advice, and

encouragement not only the social life but also in this report.

From the bottom of my heart, I am very grateful for Ph.D. Pham Nguyen Duc Hoang – Director of the

Applied Biotechnology Institute (ABI) for giving me a chance to achieve new knowledge and enhance

useful practical skills for my future career path. Additionally, this report could not be done in the best

way without any helps from Ms. Dang Hoang Quyen, who is willing to provide me with conscientious

guidance and precious lessons.

I also want to thank the office of the Biotechnology department for giving support timely in this online

semester. Last but not least, I consider myself a lucky undergraduate since I received a lot of advice

and assistance from my fellows: Ms. Yen, Ms. Thu, Mr. Hoi, Ms. Mai, Ms. Quan, Ms. Duyen, etc. in

the training program.

Ho Chi Minh City, May 16th, 2022

Ton Ngoc Minh Trang

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 TABLE OF CONTENTS
A. OVERVIEW OF THE APPLIED BIOTECHNOLOGY INSTITUTE
1. Organization structure.............................................................................................. 6
2. Functions................................................................................................................. 6
3. General missions..................................................................................................... 6

B. INTRODUCTION ............................................................................................. 7

C. BACKGROUND INFORMATION

4. Structural charateristics of ectomycorrhizal fungi .......................................................8

5. Functions of ectomycorrhizal fungi ...........................................................................10

D. ECTOMYCORRHIZAL ROOT TIPS COLLECTION

6. Handling process of Pinus kesiya...............................................................................15

7. Collection of ectomycorrhizal root tips......................................................................17

E. HYPHAL SPREAD AREAS COMPARISON BETWEEN SUILLUS BOVINUS’S STRAINS

8. Objectives .................................................................................................................21

9. Materials and chemicals preparation..........................................................................22

10. Culture procedure....................................................................................................25

11. Results and discussion..............................................................................................26

F. PERSONAL JUDGEMENT..............................................................................33

G. REFERENCE.....................................................................................................34

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A. OVERVIEW OF THE APPLIED BIOTECHNOLOGY INSTITUTE
The Applied Biotechnology Institute (ABI), certificated by the Ho Chi Minh City Department of
Science and Technology, was established on June 22nd, 2021. The main laboratory is now located on
the fourth floor, Van Dat Tower, CN8 Street, Tay Thanh Ward, Tan Phu District, Ho Chi Minh City.
1. Organization structure:
Directorate board:
• Director: Ph.D. Pham Nguyen Duc Hoang
• Vice Director: MSc Nguyen Tai Hoang
Departments:
• Mushroom Farming
• Physiology & Biochemistry
• Cryopreservation and Culture
• Taxonomy and Morphogenesis
• Molecular Biology and Genetics

2. Tasks and purposes:

 Performing primary investigation in applied Biotechnology and related areas.
 Consulting, technology transferring, and training high qualified researching human resources in
Mycology, Plant Protection, Biotechnology, and related areas according to rules.

3. General missions:
 Basic research conducted on Mycology, Plant Protection, Biotechnology and related areas.
 Highly developed skilled science and technology workers in the fields of Mycology, Biotechnology,
Plant protection, and other relevant fields.
 Stimulation of global cooperation in Mycology, Biotechnology, Plant Protection and related areas.
 Transfer of technology.

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 Time
Description
(week)
- Meeting with on-site supervisor:
1  Applied Biotechnology Institute (ABI) introduction
14/3 – 20/3  Summary of reaserch fields in ABI
 Lab safety and rules guidance
- “Modified Melin-Norkrans” medium preparation

2 - Practice of micropipetting and inoculating skills

21/3 – 27/3 - Ectomycorrhizal root tips collection and observation

- Preparing medium and tools for inoculation

- Inoculating Suillus bovinus’ strains
3-5
- Keeping track and taking pictures of the cultured samples during
28/3 – 17/4
three weeks
- Recording and analyzing data by SPSS software

- Reporting the results

6-7 - Conducting a comparison of the hyphal spread areas of Suillus
18/4 – 30/4 bovinus strains on the “Modified Melin-Norkrans” medium
- Getting comments from on-site supervisor

8–9 - Completing the report

1/5 – 14/5 - Final feedback from on-site supervisor and IU’s advisor

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B. INTRODUCTION TO

Pinus kesiya, one of the species in Pinaceae family, is observed to distribute most widely in Asian
forests and savannah, ranging from the northeast Indian state of Meghalaya to northern Thailand,
Philippines, Cambodia, southernmost China, and Vietnam: Lai Chau, Lang Son. This kind of species is
a relatively fast-developing evergreen, the coniferous tree growing up to 45 meters in height since it
possesses a straight, cylindrical bole with 15-20 meters branches and 100 centimeters diameter. It
contributes mostly to the total production of wild timber and resin in the Southeast Asian region as the
trees are regularly cultivated in plantations.
Roots - an important organ for water and nutrient absorption – can give support to enhance a symbiotic
relationship with mycorrhiza by releasing carbon compounds in the form of exudate. The most
circumstantial explanation for the term “symbiotic relationship” is that while mycobiont takes
advantage of the extensively branched hyphal web for the plant’s access to water and nutrients sources,
the plant provides carbonhydrates to maintain the growth and survival of fungi, particularly in
ectomycorrhizas (EcMs). Thanks to this relationship, EcMs can grow in the nutrient-poor environment
whereas ectomycorrhizal trees benefit from resisting soil-borne disease and enduring drought stress.
More than a hundred years have passed since Frank (1894) first developed the hypothesis relating to
the significance of the ectomycorrhizal symbiosis by predicting that mycorrhizal fungi absorbed
nitrogen from layers of soil in the forest, passed it into the trees and simultaneously obtained carbon to
make themselves more sustainable. Not until the year 1966 did Singh first note that dipterocarps, like
other trees in tropical forests, formed EcMs. Since then, the roots of multiple dipterocarps have been
performed researches on and been observed with ectomycorrhizal fungi colonies (Alexander and
Högberg 1986; Chalermpongse 1987; Hoang and Tuan 2008). Despite the plenty of accumulated
information on morphology and physiology, a shortage of reports about ectomycorrhizal fungi on roots
requires more details for this potential colonization.
In the range of this report, I have demonstrated a summary of the soil-handling process to collect
ectomycorrhizal root tips from Pinus kesiya species. After that, I conducted to compare the hyphal
spread areas of Suillus bovinus strains on the Modified Melin-Norkrans (MMN) medium.

8
C. BACKGROUND INFORMATION

4. Structural characteristics of ectomycorrhizal roots

Hartig net, the complicated fungal structure, describes a closely acquainted relationship among the
ectomycorrhizal partners. Hartig net is defined as a network of growing fungal hyphae extending to the
root, penetrating between the radial walls of the cortex’s outer cells to produce branched and closely
packed fan-like enhancement. Large surface areas are observed to exist in the individual walls of each
hyphal branch and to carry the analogous structure of transfer cells in plants. There are several recorded
hyphal features such as being coenocytic, multinucleate and consisting of mitochondria also rough
endoplasmic reticulum.

Unlike the uniform structure of the Hartig net, outside layers covering roots, the mantles, show the
diversity of anatomical and morphological characteristics that permit not only categorization based on
shape or color but also fungal genera and symbiosis species identification. To be more specific, the
typical cell arrangement patterns in parenchymatous and plectenchymatous matrices can even diagnose
at the species levels through mantle scrapings. Where multiple crucial mycorrhiza genera can be
recognized under the microscope such as Russula, Amanita and Lactarius, several unidentified
common fungal types – Piceirhiza and Fagirhiza- are classified based on their host genera – spruce and
beech, respectively.

Although being considered as the most uncontrollable component, vegetative mycelium is also the
most vital one in the ectomycorrhizal system that can both assist the root in getting access to the
surroundings and satisfies the nutrients exploration and exploitation demands for transporting to the
mantle. This function requires the unity of the mycelial network, the intrinsic fragility with the nature
of elements under the microscopic aspects and their inaccessibility. There are also prerequisites for
realistic evaluation of mycorrhizal mycelium functions and structures including using natural substrates
without additional exogenous carbon resources and maintaining the microbial community. Numerous
researches have been conducted on the ectomycorrhizal mycelia structure, extension and
differentiation. Scientists recorded two types of growth patterns. The first one originating from
colonized roots largely grows undifferentiated fan-producing hyphae that can engage the soil. These
patterns are effortlessly observed in ascomycetous ectomycorrhiza, Cenococcum geophilum and
Piloderma croceum, and some types of basidiomycetes. Meanwhile, the development of the second
hyphal type takes place continuously.

Similar to the previous type, this type of hyphae starts to grow for fan production with a velocity up to
2-3mm per day. A noticeable fefeaturef these fans is that they can gather to form compact linear organs
called “cords”, “strands” and “rhizomorphs”. These wide “vessel” hyphae possessing few or no septa

9
and the 30µm diameter are covered in thickly cytoplasmic layers. Nevertheless, instead of penetrating
the mantle, these vessel hyphae branch frequently outside and then enter it under the fan-like structure
of smaller elements, which is observed to be similar to a river delta. In spite of being considered a
disordered tuft-like structure in detached roots, these hyphal aggregates are still recorded in the
complete system to stick basipetal to the mycorrhizal root’s mantle or the rootlets’ clusters. It appears
to be the main and only contact between the mantle and soil.

Mycelial cords penetrating the mantle are the channel that assist the colonized rootlets in getting access
to soil resources. By observing representatives of several crucial genera such as Russula, Suillus,
Amanita, etc., scientists give strong support for the idea that the mantle plays an important role in
storing than absorbing nutrients. Whereas these features can benefit the obtained resources
maintenance, there are some noticeable drawbacks for an absorptive function. Besides, the hydrophobic
ability is dependent on the fungal categories; for example, the mantle formed by Cenococcum and
Thelephora seems not to be more hydrophobic than others. At the end of the mycelial system, the
hyphae move forward as a broad front that helps it achieve the leading efficiency of exploration in the
system with patchily allocated resources. The areas witnessing the high-rate proliferation are termed
“patches”’, which are also known as nutrient-supplied places that give support for fungi to forage
intensively. Via researches were conducted in 1991 and 1995 by Carleton & Read and Beding & Read,
respectively, it is notable that such patches can be conformed and exists under the scattered units after
the front go through. Scientists performed the experiment by collecting organic remnants from the
fermentation horizon (FH) of forest soil and placing them in the development directions of the
advancing front. During the experiment, if there is any colonization of FH material, it might say that
the FH region is engaged by a considerable ratio of fungal biomass.

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5. Functions of ectomycorrhizal fungi
a. Complicated organic resources mobilization

Multiple outstanding researches performed by Lindeberg (1944), Norkrans (1950) and Lundeberg
(1970) denied the ability to use the complicated organic resources of the major nutrients nitrogen and
phosphorus. Besides, Melin and Harley’s laboratory studies made this view more certain by proving
that the plant’s capability to absorb phosphate and ammonium can be enhanced thanks to
ectomycorrhizal colonization. However, the qualitative nature of materials still maintains as a question
until now.

The fact that nitrogen accretion on the surface organic residues results from the small proportion of
mineralization is easy to be observed during typical ectomycorrhizal trees. The plants’ demands for
nitrogen are 10 times higher than those for phosphorus, which makes nitrogen become the
indispensable growth nutrient. Since the roots selectively grow in the FH layers of soil with the greatest
nitrogen mobilization, it is not uncooperative to believe in Frank’s conclusion on the collection of
mycorrhizal roots in organic remnants for nitrogen mobilization.

Complicated organic polymers cannot qualitatively represent the substrate that fungi contact with in
nature. On the other hand, under the non-sterile condition, natural substrates composes of microbial
population mixture may interfere with the quantification process of ectomycorrhizal fungi. Therefore,
we must combine methods for fungal biochemical potential examination with complicated natural
substrates to achieve the expected results.

A laboratory experiment is performed by using soluble protein as high molecular weight nitrogenous
substrates (Melin 1925; Fontana, Read & Abuzinadah 1986). As a result, these “protein fungi”
willingly take advantage of such polymers as the only nitrogen source. When these fungi are cultured
and provided with protein as the only nitrogen source, the ratio of this element, usually under the form
of amino compounds (Finlay 1988; Read 1989), is transported to the host (Abuzinadah and Read, 1986,
1989). When being cultured with the only nitrogen source and supplied with carbon like the host in
nature, “protein fungi” only deliver ammonium in case of exogenous carbon’s exhaustion.
Consequently, the hypothesis of considering mineralized forms as the sole or even the most crucial
nitrogen source is not appropriate.

b. Acid carboxyproteinase production

“Protein fungi” do contribute to the acid carboxyproteinase producing process; although their amount
of expressed protease activity in the specific standard condition is lower if compared to those of ericoid
fungi, it may be related to some known ecological features. For instance, during the observation,
Suillus luteus and Suillus grevillei are among the most active protease producers. On the contrary,

11
Pisolithus tinctorius, a little enzyme-producing fungus in mineralizing environment, and Cenococcum
geophilum, a fungus colonizing hosts on soils in an alpine environment, possess the ineffective ability
to produce protease. Besides, the research on nitrogen transport from fungus to the host corresponds to
the enzyme assays with the descendent order Hebeloma crustuliniforme, Amanita muscaria, Paxillus
involutus.

Figure 1: Relationship between proteinase activity of typical ectomycorrhizal fungi grown under the same
conditions. The enzyme activity is inhibited when pH above 4.0.

There is a variety of ectomycorrhizal sensitivities to culture conditions for enzyme activity production;
therefore, we have to treat the experimental screening results in a careful way. The ectomycorrhizal
fungi’s capability to satisfy the nitrogen demands by absorbing the proteins of plant and microbial
remnants of the host roots is no more doubted. Nevertheless, whether this ability is maintained when
getting access to natural substrates is still a question. As these fungi strongly proliferate over the FH
materials, which expresses an ideal connection, the fungal structural capabilities for natural substrates

12
are more clear. The nitrogen concentration increase in the host foliage is noted to have a relationship to
the conformation of mycelial patches from FH zone. Because of this, an experiment is performed to
examine the functions of the exploitative growth methods.

Researchers started to radioactively label carbon used to feed the mycorrhizal plants’ shoots so that the
mycelia of Suillus bovinus could extend to form “patches” in clusters of organic compounds from pine
soil FH horizon. It took a short duration approximately 40 days for these patches’ carbon distribution to
occur.

Figure 2:
(a) Suillus bovinus mycelium with fermentation horizon organic matter (FH) trays at different colonization phase. (b)
Autrodiograph of mycelium in (a) after supplying 14CO2. Carbon distribution to the tray colonised is less than 40 days.

Initially, sturdy colonization and patch conformation are observed via consecutive pictures and
microscopy but soonly, there are decreases and transforms of the color of ectomycorrhizal mycelium
and saprotrophic fungi’s capacious sporulation (Fig 8). This pattern shows that the exploitative stage
also possesses a time restriction for the specific resources mobilization.

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 Figure 3: . Developmental stages of the Suillus
bovinus mycellium from mycorrhizal roots of P
sylvestris to colonize homogeneous peat and trays
of fermentation horizon organic matter (FH) a)
Day 0 b) Day 14 c) Day 28 d) Day 42

Through the examination of the content of major nutrients, nitrogen, phosphorus and potassium before
and after colonization of the FH material, Suillus bovinus exploitation contributed to the export of these
substances. To be more specific, during 40 days, the concentration of such elements went down by 23,
22 and 30%, respectively, which happens contemporaneously to the carbon import of patches. Leake
(1995) illustrated the estimated values of nitrogen and phosphorus released by Suillus bovinus is 32 and
33%, respectively. This evidence was not strong enough for microbial ammonification to explain the
pattern of nitrogen depletion but did support the relationship between the exploitation of organic
remnants and ectomycorrhizas.

The fact that the FH remnants’ colonization of Suillus bovinus export phosphorus under the form of
phosphatase happens when exposed to organic sources. Because there is a large number of phosphorus
present in the root of forest soils, the phosphatase form will have great importance. Phosphorus, in
organic form in mull humus soils with the ability to maintain the amount of nitrifying bacteria in the
root, might become the principal growth restricting factor. Phosphatase activities may be the
indispensable deciding influence on ecosystem productivity and in the wider boreal forest context,
where nitrogen saturation is a result of pollutant inputs (Aber et a1., 1989), and the demand for further
research of these enzymes and enhanced characterization of the substrates has been emphasized.

14
Thanks to the laboratory investigation of ectomycorrhizal fungal activities, a structural background for
the selective allocation pattern of ectomycorrhizal roots in the organic horizons is established. This is
the zone that nitrogen and phosphorus mobilization happens most actively, with periods of elements’
net immobilization linked with freshly fallen litter, as saprotrophs import them to facilitate carbon
utilization (Berg & Söderström 1979), followed by the mineralization phase in FH layer. Thus, it seems
that there is involvement between the ectomycorrhizal system and the large amount of carbon
distribution, non-growth limiting resources, for absorbing those resources, nitrogen and phosphorus,
restricting growth factors in natural ecosystems. Besides, carbon also plays a crucial role both in plants
nutrients and in sustaining soil ecosystems activities

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 D. ECTOMYCORRHIZAL ROOT TIPS COLLECTION

6. Handling process of Pinus kesiya

a. Materials

Materials Quantity

Plastic zipper bag 60

Tray 4

Plastic plate 4

Brusher 4

Forceps 2

Branch-cutting scissors 1

Cellophane tape 1

Marker 1

Sample: 15 pine trees of Pinus kesiya species collected from the sampling plant plots in Vietnamese
central highlands.

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 b. Procedure

First, we went to the sampling plant plots in Vietnamese central highlands and chose the most
revitalized and transportable pine trees belonging to Pinus kesiya species. We kept about 50g of soil for
further research. After collecting 15 pine trees, we commenced dividing them by branch-cutting
scissors into 4 parts: roots, trunks, branches and leaves, then put each partinto plastic zipper bags for
storage to weigh hydrated biomass before drying them. The root parts of pine trees were kept in the soil
in the plastic zipper bags so that they can maintain their humidity to ensure that there weren’t any
errors when we weigh the roots’ hydrated biomass.

Figure 4: Obtained pine leaves before being dried (left) and after being dried (right)

Figure 5: The trunk part (left) and the branches (right) of a pine tree

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 7. Collection of ectomycorrhizal rootlets

After that, we collected all of the main roots from the soil to weigh for biomass and the remaining soil
mixture was scattered in a tray for obtaining ectomycorrhizal rootlets. Next, we gently digged the soil
up by hand to seek ectomycorrhizal roots’ tips in a careful way. During the search, it was possible for
us to mistake grassroots for pine rootlets if we did not pay attention to their differences.

Figure 6: Soil was scattered before being digged up for

searching ectomycorrhizal rootlets’ tip

Figure 7: The selecting process of ectomycorrhizal rootlets from the soil mixture. The plastic plates contained pine roots
and ectomycorrhizal roots’ tips while the brackets were used to keep grassroots and residues.

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19
The difference between grass roots and pine rootlets is shown in the following table.

Grass roots Pine rootlets

Thin, flat shape Thicker, round shape
Light color Dark color
Odourless Wooden scent

After collecting all of ectomycorrhizal rootlets, we put them into a plastic plate with little water (Figure
9) to maintain the humidity of rootlets and to remove all of the soil remnants to make it possible to
clearly observing these rootlets under the microscope.

Figure 9: Plates contained pine rootlets and ectomycorrhizal root tips soaked
in water
These
ectomycorrhizal root tips usually share several traits, which are branch divisions, bulges and hyphae
conformation, compared to the normal ones. In the structure aspect, the mantle layer in
ectomycorrhizas covers host roots, which suppress their hair root development via the hormone
interactions between fungi and plants. This phenomenon is made up of the extension of extraradical
hypha from the mantle into the soil to enhance the surface area of the colonized root. This part of
ectomycorrhiza functions as a nutrients, carbon and water transport unit by spreading noticeable
distances and maintaining a large accessed area with soil. These hyphae can form an aggregate
arrangement called rhizomorph relating to the degree of rhizomorph organization and the transport
rates of nutrition. These are explanations for the reason why ectomycorrhizal root tips differ from the
normal ones. However, because the ectomycorrhizal mantle usually expresses characteristic features

20
ectomycorrhizal root tips. Some types possess yellow or black colors while others are white or dark
brown. The following are multiple photographs of ectomycorrhizal root tips through the naked eyes.

21
Then, we started to observe their morphology and the number of root tips under the microscope and
note them down. As we see in Figure 10, ectomycorrhizal root tips had a tendency to bulge more than
the normal tips to extend the surface area for nutrient absorption. These tips were also covered by a
fungal sheath – the mantle with multiple colors such as black, dark brown (picture A) or white (picture
B). Using the microscope with high resolution can assist us in observing hyphae surrounding the
mantle layers. Besides, there are a variety of ectomycorrhizal root tips’ shapes existing in nature.

Figure 10: Ectomycorrhizal root tips observed under the microscope

Figure 11: Various shapes of ectomycorrhizal root tips illustrated by drawings

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E. HYPHAL SPREAD AREAS COMPARISON BETWEEN SUILLUS BOVINUS’S
STRAINS

8. Objectives
Suillus genus is observed to widely distribute all over natural pine forests developing in the Nothern
Hemisphere. Species in this genus have a specialized mycorrhizal relationship to the plants of
Pinaceae family making it becomes different from most of other ectomycorrhizas. Thanks to the
fast growth and easy culturing procedure, Suillus species are often focused by many scientists for
fungal researches.
The slimy and sticky cap cuticle when moist, the existence of cystidia - dark clustered sterile cells,
cinnamon or chocolate brown in spores, and strictly mycorrhizal relationships with members of the
Pinaceae, particularly with members of the genera Pinus are the outstanding characteristics of
Suillus.
Until now, there are approximateky 98 species in Suillus genus are reported over the world. In
1940, Suillus bovinus, Suillus grevillei and Suillus luteus were first announced to belong to Boletus
genus while in 1959, these 3 species were divided into Suillus genus and recorded to be an edible
species.
Generally, Suillus genus are one among the ectomycorrhizas plays an important role in nutrient,
economy, etc., contributes to the natural diversity and provides the science with sustainable, steady
and lasting values.
In this part, we conducted an experiment to culture 10 strains in Suillus bovinus with Modified
Melin-Norkrans (MMN) agar medium during 3 weeks. After that, we analyzed the obtained data for
comparison between these 10 strains to see which strains do have the largest spreading areas.

23
 9. Material and chemical preparations

a. List of materials and chemicals

 Materials

Materials Quantity
Petri dish 50
Duran bottle 4
Surgical blade 3
Surgical knife handle 3
Hole-punching stick 3
Graduated cylinder 2
Tools rack 1
Small gas burner 1
Laminar flow box 1
Precision scales 1
Digital scale 1
Micropipette 1
Pipette tip 1
Autoclave 1
Magnetic stirrer 1
Stir bar 1
pH meter 1

Culturing samples: 10 strains of Suillus bovinus are prepared to be cultured in the Modified Morkran
for observation and area comparison.

 Chemicals

Chemicals Quantity
(NH4)2HPO4 stock 500µg
KH2PO4 stock 1000µg
MgSO47H2O stock 300µg
CaCl2Ÿ2H2O stock 100µg
NaCl stock 50µg
24
 FeCl3Ÿ6H2O (FeEDTA) 40µg
Glucose (D-+-glucose) 20g
Malt extract 6g
Thiamine HCl 0.2µg
Distilled water 2L
Agar powder* 30g
Ethanol 70 500mL

 Calculation of the amount of FeCl36H2O and Thiamine HCl

i. FeCl3Ÿ6H2O (FeEDTA)
FeCl3Ÿ6H2O is prepared with the concentration: C = 2g/500mL
Here, 40µg of FeCl3Ÿ6H2O is required for 2 liters of MMN medium. We can set up a calculation:
2000µg  500mL

40µg  x mL

From that, the amount of FeCl3Ÿ6H2O is needed for 2 liters of MMN medium is:

40 µg ×500 mL
x= = 10mL
2000 µg

 We need 10mL of FeCl3Ÿ6H2O to concoct 2 liters of MMN medium

ii. Thiamine HCl

The concentration of Thiamine readily prepared in the laboratory is: C = 100 µg/mL
Because only 0.2µg is necessary for 2 liters of MMN medium, we have a formula:
100µg  1mL
0.2µg  x mL
We can calculate:
0.2 µg ×1 mL
x= = 2mL
100 µg
 We need 2mL of Thiamine for 2 liters of MMN medium

*
With the ruled amount of 15g/L

25
 a. Medium preparation
Step 1: Measure 2 liters of distilled water with a graduated cylinder
Step 2: Gently pour all of distilled water into a big plastic bottle with a stir bar in the bottom.
Step 3: Place the plastic cup on the magnetic stirrer and set the speed of 300-500rpm per minute
Step 4: Weigh 500µg of (NH4)2HPO4 stock, 1000µg of KH2PO4 stock, 300µg of MgSO47H2O stock,
100µg of CaCl22H2O stock and 50µg of NaCl stock.
Step 5: Pour all of these stock into the bottle with distilled water with the order: (NH 4)2HPO4 stock -
KH2PO4 stock MgSO47H2O stock - CaCl22H2O stock - NaCl stock
Step 6: Use a cylinder to measure 10mL of FeCl36H2O and pour into the plastic bottle.
Step 7: Continue to weigh 20g Glucose (D-+-glucose) and 6g Malt extract; then, add them to the plastic
bottle.
Step 8: Use a micropipette to suck 4mL of Thiamine HCl and release to the mixture.
Step 9: Measure pH of the medium by using pH measuring machine. The required pH is 5.8
Step 10: Divide the medium into 4 Duran bottles, each containing 250mL of medium
Step 11: Weigh 7.5g agar powder and add it into every Duran bottle.
Step 12: Autoclave these bottles with the temperature 121oC for 15 minutes

b. pH measurement

Before using pH measuring machine, we need to calibrate it. First, press “stdby” button to start the
meter. Next, carefully remove the glass electrode, which is very fragile, from the glass storage bottle.
To release the filling hole, twist the blue ring at the top of the electrode. Use distilled water to wash the
electrode and put it into the pH 4 calibration buffer, press “std” button and wait for the reading number.
After that, press “std” button again. Wash the electrode as earlier, put it into the pH 7 buffer, press
“std”, wait for the reading number, press “std” button one more time. Repeat one more time with the
pH 10 buffer.

To measure the pH of the MMN medium, re-wash the electrode and put it into the medium but do not
allow the electrode to touch the sides or the bottom of the bottle. The required pH is 5.8; therefore, if
the reading number is below 5.8, we can correct it by adding 1M NaOH or if we overshoot, add a few
drops of 1M HCl. When we finish, wash the electrode gently, place it in the glass storage bottle and
close the blue hole. Turn the machine off by pressing “stdby” button.

26
 10. Culture procedure
Step 1: After autoclaving, all of MMN medium bottles, cool them down before pouring it to Petri
dishes
Step 2: Turn on UV light mode in the laminar flow box to sterilize for 30 minutes. Do not open the
glass door during this process and researchers should leave the room
Step 3: Next, prepare 50 Petri dishes for 10 Suillus bovinus strains with 5 dishes per type and culturing
tools such as three hole-punching sticks, three surgical knives, a tool rack, a gas burner and an ethanol
70o glass bottle.
Step 4: After finishing the sterilized process with UV light, open the glass door and turn the fan mode
on for 30 minutes
Step 5: Open the glass door, sterilize all of the Petri dishes and necessary tools with an ethanol 70 o
sprayer before put them into the flow box
Step 6: Until the medium cooled down, pour it into each Petri dish with a sufficient amount. Wait until
it becomes dried and solidified to culture with fungi.
Step 7: Dip all of the tools in an ethanol glass bottle before flaming them with the gas burner. Wait til
them cool down.
Step 8: Pick an agar sample dish of one strain, and unseal it. Partly open its lid in a careful way and try
to keep the dish inside the area of the laminar flow box.
Step 9: Use a hole-punching stick to punch as many holes in the area with hyphae. Close its lid and put
the dish aside.
Step 10: Dip the stick in the ethanol glass bottle and flame it with the gas burner
Step 11: Before culturing, label the dates, name of strains and researchers also number from 1 to 5 on
the edge of each five Petri dishes.
Step 12: Open the early sample dish’s lid and use a surgical knife to take one circle fragment out, try to
bisect it to reduce the spreading time of hyphae.
Step 13: Put the remaining hyphae part on a labeled agar plate with the hyphae towards the lid.
Step 14: Close the lid and seal the dish carefully. Dip the knife into the alcohol bottle and flame it on
gas burner. Put it back to the rack.
Step 15: Repeat all of the steps of this procedure to 10 strains with 5 dishes per one.
Step 16: Wrap the inside of the plastic box with foil so that it can cover all aspects of the box. Put all of
the cultured Petri dishes into the box. Store it in the comfortably cool place with light deprivation.
Step 17: Take pictures of 50 dishes every week.

27
 11. Results and discussions
We chose 10 strains of Suillus bovinus and numbered for each type, which are shown in the below
table.
Strains names Identification numbers
SB1 1
SB2 2
SB3 3
SB4 4
SB5 5
SB6 6
SB7 7
SB8 8
SB9 9
SB10 10

The recorded culture date was 31st March, 2022 with 50 Petri dishes, 5 dishes for each strain. After one
week, we would take pictures to observe the spreading areas of each dish. The first week started from
31st March, 2022 to 7th April, 2022. The next two weeks began from 8 th April, 2022 to 14st April, 2022
and 15th April, 2022 to 21st April, 2022, respectively. The following are all of the photographs and data
captured, measured and calculated during the very first week.

28
a. First week results

SB1 - 07.04.22 SB3 - 07.04.22

SB2 - 07.04.22

SB4 - 07.04.22 SB5 - 07.04.22 SB6 - 07.04.22

SB7 - 07.04.22 SB8 –07.04.22 SB9 – 07.04.22

SB10 –07.04.22
 b. Second week results

SB1 - 15.04.2022 SB2 -15.04.2022 SB3 - 15.04.2022

SB4 - 15.04.2022 SB5 - 15.04.2022 SB6 - 15.04.2022

SB7 - 15.04.2022 SB8 - 15.04.2022 SB9 - 15.04.2022

SB10 - 15.04.2022
c. Third week results

SB1 - 21.4.2022 SB2 - 21.4.2022 SB3 - 21.4.2022

SB4 – 21.4.2022 SB5 – 21.4.2022 SB6 – 21.4.2022

SB7 – 21.4.2022 SB8 – 21.4.2022 SB9 – 21.4.2022

SB10 – 21.4.2022
The data analysis was proceeded with 3 petri dishes per strain to see which strain has the most
spreading areas among 10 ones. Assuming the test is normally distributed, we use ANOVA one way
with Duncan test were applied to calculate and classify the data during every single weeks.

DUNCAN CLASSIFICATION
WEEK 1 WEEK 2 WEEK 3
(31 MARCH, 2022 –
ST
(8 MARCH, 2022 –
TH
(15 MARCH, 2022
TH

7TH APRIL, 2022) 14TH APRIL, 2022) – 21ST APRIL, 2022)

SB1 0.59ab ± 0.26 1.64b ± 0.02 3.37cd ± 0.01
SB2 0.29d ± 0.02 0.61c ± 0.52 1.54e ± 1.41
SB3 0.28d ± 0.06 1.00c ± 0.11 2.22de ± 0.26
SB4 0.33cd ± 0.03 0.86c ± 0.11 2.33cde ± 0.13
SB5 0.46bcd ± 0.08 1.65b ± 0.16 3.32cd ± 0.15
SB6 0.54ab ± 0.04 1.98b ± 0.28 3.18cd ± 0.72
SB7 0.31cd ± 0.02 1.66b ± 0.14 3.50c ± 0.34
SB8 0.47bc ± 0.04 1.66b ± 0.14 4.88b ± 0.41
SB9 0.32cd ± 0.06 0.61c ± 0.33 1.64e ± 1.19
SB10 0.65a ± 0.06 3.15a ± 0.09 6.67a ± 0.34

During the first week, there are 6 strains - SB2, SB3, SB4, SB5, SB7 and SB9 (µ2 = µ3 = µ4 = µ5 = µ7
= µ9) – appear in subset 1, which means that 6 strains are not significantly difference from each other.
In the second subset, we can see that strains SB4, SB5, SB9 and SB7 also exist with SB8; therefore,
there is not any significant difference between them (µ4 = µ5 = µ7 = µ8 = µ9). However, if we
compare SB2 and SB3 to SB8, they are significantly different because they are not observed to be in
the same subset resulting from the difference in means (µ2 = µ3 ≠ µ8). Apart from the two previous
strains: SB4 and SB5, the third subset witnesses the next emerge of SB6 and SB1 proving that they are
not significantly different (µ1 = µ4 = µ5 = µ6). As we mentioned before, between the subsets, there are
no significant differences. Thus, the strains SB2 and SB3, SB8, SB6 and SB1 are significantly
different since they only exist in first, second and third subsets (µ1 ≠ µ2 ≠ µ3 ≠ µ6 ≠ µ8), respectively.
The last subset are recorded to have 3 strains, SB6 and SB1, already seen in the previous one, and
SB10 (µ1 = µ6 = µ10). Due to the absence in the three remaining subsets, SB10 strain is considered as
the most significantly different one and also the group have the highest mean (µ10 = 0.65 ± 0.06) while
SB2 (µ2 = 0.29 ± 0.02) and SB3 (µ3 = 0.28 ± 0.06) are recorded to possess the smallest means.
In the next second week, strains SB2, SB3, SB4 and SB9 are not significantly different because of
their presences in subset 1 (µ2 = µ3 = µ4 = µ9) . Besides, there is not any strains belong to both subset
1 and 2 meaning that all of these strains are significantly different from each other. Additionally, within
the subset 2, strains SB1, SB5, SB6, SB7 and SB8 do have significant difference (µ1 = µ5 = µ6 = µ7 =
µ8). The strains SB10 continue to be significantly different from all other and to have the highest mean
(µ10 = 3.15 ± 0.09). A noticeable point in this week’s results is that between those, there is no strains
belongs to both subsets.
The final week does not witness significant difference the among SB2, SB3, SB4 and SB9 strains (µ2 =
µ3 = µ4 = µ9). Next, SB3 and SB4 strains still present in the subsets 2 with SB1, SB5 and SB6 so there
is no significant difference between them (µ1 = µ3 = µ4 = µ5 = µ6). Nevertheless, if we compare SB2
and SB9 to A6112, SB5 and SB1, they are significantly different because they are not seen to be in the
same subset resulting from the difference in means. The third subset witnesses the existence of SB7
beside SB1, SB4, SB5 and SB6 proving that they are not significantly different (µ1 = µ4 = µ5 = µ6 =
µ7). The absence in the remaining subsets leads SB8 and SB10 to be the most significantly different
ones and also the strain have the highest mean (µ10 = 6.67 ± 0.34) is SB10.

Average spreading areas of 10 Suillus bovinus strains

in 3 weeks
8

0
A27302 (1) A09108 (2) A05113 (3) A07301 (4) A04127 (5) A06112 (6) A21108 (7) A08201 (8) A06115 (9) A06116
(10)

WEEK 1 WEEK 2 WEEK 3

33
As we can see in the bar chart, there are some high values of standard deviations among these Suillus
bovinus strains such as strain SB2, SB6, SB9 during both week 2 and 3. However, the growth in value
of standard deviations ranging from ± 0.26 to ± 1.41 are witnessed the final week better than two
previous ones. Nevertheless, there are a few exceptions decrease slightly in this value such as in strain
SB1 goes down from ± 0.26 to ± 0.01. The higher standard deviations are, the less reliable our
experiment is. This phenomenon can be clarified by techniques during our culturing and measuring
procedure. To be specific, during the culturing process, there are some differences in medium we
poured out. The medium layers may be thinner than the required amount leading to the contraction by
time, which causes the errors in the measurement between dishes in 3 weeks. Moreover, in case we do
not upside down the dishes after culturing 1 – 2 days, water drops could appear on the lids and obstruct
the the fungi so we cannot observe it clearly via photographs to measure it in an accurate way. This can
be solved by placing a cup of boiled water on the lid so that it would evaporize these drops.
Additionally, using artisanal techniques to measure the spreading areas is not always give out the exact
results due to faults when we draw by hands without any assistance from other tools.

34
F. PERSONAL JUDGEMENT

During this semester internship, I have achieved a variety of new knowledge about mycorrhiza in
general and about ectomycorrhiza in particular. Specifically, I can clarify by myself the questions
relating to ectomycorrhizal conformation, structure and characteristics features. Besides, the
differentiation between ectomycorrhizal pine rootlet tips and normal ones by naked eyes also the
observation those types of rootlets via a microscope becomes more easily to be proceeded. Previously,
I had been trained how to divide pine trees into seperated parts for weighting biomass and remove dirts
from the rootlets tips without the mantle lost. With the conducted experiment, there are many things
that I have learned through it:
+ Chemicals, media and tools preparation
+ Methods for using laminar flow box, autoclave machines, magnetic stirrer, micropipette, etc.
+ Safe culture techniques
+ Logically experimental designs and data arrangements
+ Photographic skills
+ Analytic skills of statistics by using software SPSS

Above all, I really appreciate my instructors for guiding me to develop soft skills such as teamwork,
communication, observation and evaluation skills beside laboratory techniques, which assists me a lot
in my internship course. Last but not least, it is impossible not to mention the constructed
responsibility and honesty during the practice time at the laboratory

35
G. REFERENCES

Bledsoe, C. S. (1992). Physiological ecology of ectomycorrhizae: Implications for field application.

Mycorrhizal Functioning: An Integrative Plant-Fungal Process.

Garbaye, J., & Bowen, G. D. (1989). Stimulation of ectomycorrhizal infection of Pinus radiata by some

microorganisms associated with the mantle of ectomycorrhizas. New Phytologist, 112 , 383–

388.

Lakhanpal, T. N. (2000). Ectomycorrhiza—An overview. Mycorrhizal Biology, 101–118.

Marks, G. C. (2012). Ectomycorrhizae: Their ecology and physiology. Elsevier.

Müller, T., Avolio, M., Olivi, M., Benjdia, M., Rikirsch, E., Kasaras, A., Fitz, M., Chalot, M., & Wipf,

D. (2007). Nitrogen transport in the ectomycorrhiza association: The Hebeloma

cylindrosporum–Pinus pinaster model. Phytochemistry, 68 , 41–51.

Rai, M., & Varma, A. (2010). Diversity and biotechnology of ectomycorrhizae (Vol. 25). Springer

Science & Business Media.

Stocchi, V., Bonfante, P., & Nuti, M. P. (1995). Biotechnology of ectomycorrhizae: Molecular

approaches. Springer.

36
TABLE ILLUSTRATING SPREADING AREAS OF 10 SUILLUS BOVINUS STRAINS

 First week’s results (31st March, 2022 - 7th April, 2022)

AREAS AFTER 7 DAYS

STRAINS PLATES
(cm2)
1.1 0.290428655
1.2 0.682560188
SB1 1.3 0.789312977
1.4 0.445096888
1.5 0.552319436
2.1
2.2
SB2 2.3 0.305578391
2.4 0.260364063
2.5 0.290663535
3.1
3.2 0.236523782
SB3 3.3 0.254374633
3.4 0.355725191
3.5 0.342571932
4.1 0.295243688
4.2 0.364180857
SB4 4.3 0.321785085
4.4 0.416324134
4.5 0.348326483
5.1 0.547269524
5.2 0.428537874
SB5 5.3 0.407751028
5.4 0.360657663
5.5 0.399295361
6.1 0.509923664
6.2 0.52472108
SB6
6.3 0.590135056
6.4 0.483382267

37
 6.5 0.395067528
7.1 0.322019965
7.2 0.290428655
SB7 7.3 0.321785085
7.4 0.294773928
7.5 0.353611274
8.1 0.332119789
8.2 0.378156195
SB8 8.3 0.519201409
8.4 0.446623605
8.5 0.452260716
9.1 0.389665297
9.2 0.30604815
SB9 9.3 0.278684674
9.4 0.333059307
9.5 0.328831474
10.1 0.639459777
10.2 0.720258368
SB10 10.3 0.596711685
10.4 0.601996477
10.5 0.693012331

38
 Second week’s results (8thApril, 2022 - 14th April, 2022)

7.1 AREAS AFTER 14 DAYS

1.80248924
STRAINS PLATES
7.2 (cm )
2
1.531464464
SB7 7.3
1.1 1.653134814
1.627777132
7.4
1.2 1.829940677
1.617889962
7.5 1.534023497
SB1 1.3 1.664883099
8.1 2.251948354
 1.4
8.2 1.616959404
0.731883215 Third
SB8 1.5
8.3 1.496335931
2.165755496 week’s
8.4
2.1 2.427939979 results
AREAS AFTER 21 DAYS
STRAINS PLATES
8.5
2.2 2.139467256
(cm2) (15st
9.1 0.959637083
SB2 2.3
1.1 1.208444806
3.369689877 April,
9.2 0.311038734
SB9 2.4
1.2
9.3 0.234267768
3.368519602
0.556356869 2022 –
SB1 2.5
9.4
1.3 0.233104571
0.27288589
3.385839672 21st
9.5
3.1 0.459229964 April,
1.4 3.369923932
10.1 3.195068047 2022)
3.2
1.5 1.086076538
3.145231129
10.2 3.043619867
SB3
SB10 3.3
2.1
10.3 0.879725486
3.093637315
3.4
2.2
10.4 1.024543445
3.196463883
SB2 10.5
3.5
2.3 3.334651623
0.589856927
3.093739029
4.1
2.4 0.921251599
1.153891164
4.2
2.5 0.938001628
0.362434172
7.1 3.863897016
SB4 4.3
3.1 0.732581133
7.2 3.181158572
SB7 4.4
3.2
7.3 2.503920421
3.443417203
SB3 4.5
7.4
3.3 0.848435501
4.407723815
2.177296665
7.5
5.1
3.4 3.21591574
1.844364313
1.991691047
8.1
5.2 4.61263897
1.552867279
3.5 1.743241662
8.2
SB5 5.3
4.1 1.567639874
2.39812756
SB8 8.3 4.550731422
5.4
4.2
8.4 1.653134814
2.413809245
5.339379754
SB4 5.5
8.5
4.3 1.590671164
4.756465769
2.179520187
9.1
6.1
4.4 2.610415448
1.991392346
9.2
6.2 1.99953189
1.695591485
4.5 2.153774137
SB9 9.3 0.314569924
SB6 6.3
5.1
9.4 2.251948354
3.42293739
6.4
5.2
9.5 1.946958241
3.145231129
SB5 10.1
6.5
5.3 7.026331188
1.43747819
3.378115857
10.2
5.4 6.357519017
3.494558221
SB10 10.3
5.5
10.4 3.331538912
6.627735518
6.1
10.5 3.046342891
6.800702165
6.2 39 2.534932709
SB6 6.3 3.958104154
6.4 3.351667642
6.5 2.529666472
40
 AFTER USING SPSS SOFTWARE TO ANALYZE THE OBTAINED DATA
 First week’s results (31st March, 2022 - 7th April, 2022)

Descriptives
SEVENDAYS

N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum

Lower Bound Upper Bound

SB1 3.00 .59 .26 .15 -.07 1.24 .29 .79

SB2 3.00 .29 .02 .01 .23 .34 .26 .31
SB3 3.00 .28 .06 .04 .12 .44 .24 .36
SB4 3.00 .33 .03 .02 .24 .41 .30 .36
SB5 3.00 .46 .08 .04 .27 .65 .41 .55
SB6 3.00 .54 .04 .02 .44 .65 .51 .59
SB7 3.00 .31 .02 .01 .27 .36 .29 .32
SB8 3.00 .47 .04 .02 .37 .57 .45 .52
SB9 3.00 .32 .06 .03 .18 .47 .28 .39
SB10 3.00 .65 .06 .03 .50 .80 .60 .72
Total 30.00 .42 .15 .03 .37 .48 .24 .79

ANOVA
SEVENDAYS

Sum of Squares df Mean Square F Sig.

Between Groups .505 9 .056 6.144 .000

Within Groups .183 20 .009
Total .688 29

41
 SEVENDAYS

STRAINS N Subset for alpha = 0.05

1 2 3 4

A05113 3 .28

A09108 3 .29

A21108 3 .31 .31

A06115 3 .32 .32

A07301 3 .33 .33

Duncana A04127 3 .46 .46 .46

A08201 3 .47 .47

A06112 3 .54 .54

A27302 3 .59 .59

A06116 3 .65

Sig. .05 .08 .15 .19

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

42
  Second week’s results (8th April, 2022 – 14th April, 2022)

ANOVA
Descriptives
FOURTEENDAYS
FOURTEENDAYS
Sum of Squares df Mean Square F Sig.
N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum
Between Groups 15.83 9.00 1.76 31.79 .00
Lower Bound Upper Bound
Within Groups 1.11 20.00 .06
SB1 3.00 1.64 .02 .01 1.58 1.70 1.62 1.66
Total 16.93 29.00
SB2 3.00 .61 .52 .30 -.69 1.90 .26 1.21
SB3 3.00 1.00 .11 .06 .73 1.26 .88 1.09
SB4 3.00 .86 .11 .07 .58 1.15 .73 .94
SB5 3.00 1.65 .16 .09 1.25 2.06 1.55 1.84
SB6 3.00 1.98 .28 .16 1.29 2.67 1.70 2.25
SB7 3.00 1.66 .14 .08 1.33 2.00 1.53 1.80
SB8 3.00 1.66 .14 .08 1.33 2.00 1.53 1.80
SB9 3.00 .61 .33 .19 -.20 1.42 .31 .96
SB10 3.00 3.15 .09 .05 2.93 3.36 3.04 3.20
Total 30.00 1.48 .76 .14 1.20 1.77 .26 3.20

43
 FOURTEENDAYS

STRAINS N Subset for alpha = 0.05

1 2 3 4

A09108 3.00 .61

A06115 3.00 .61

A07301 3.00 .86

A05113 3.00 1.00

A27302 3.00 1.64

Duncan a
A04127 3.00 1.65

A21108 3.00 1.66

A08201 3.00 1.66

A06112 3.00 1.98

A06116 3.00 3.15

Sig. .08 .12 1.00

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

44
  Third week’s results (15th April, 2022 – 21st April, 2022)

Descriptives
TWENTYONEDAYS

N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum

Lower Bound Upper Bound

SB1 3 3.37 .01 .01 3.35 3.40 3.37 3.39

SB2 3 1.54 1.41 .81 -1.95 5.03 .36 3.09
SB3 3 2.22 .26 .15 1.58 2.87 1.99 2.50
SB4 3 2.33 .13 .08 2.01 2.66 2.18 2.41
SB5 3 3.32 .15 .09 2.95 3.69 3.15 3.42
SB6 3 3.18 .72 .42 1.39 4.97 2.53 3.96
SB7 3 3.50 .34 .20 2.64 4.35 3.18 3.86
SB8 3 4.88 .41 .24 3.87 5.90 4.55 5.34
SB9 3 1.64 1.19 .69 -1.31 4.60 .31 2.61
SB10 3 6.67 .34 .19 5.83 7.51 6.36 7.03
Total 30 3.27 1.60 .29 2.67 3.86 .31 7.03

ANOVA
TWENTYONEDAYS

Sum of Squares df Mean Square F Sig.

Between Groups 65.602 9 7.289 16.512 .000

Within Groups 8.829 20 .441
Total 74.431 29

45
 TWENTYONEDAYS

STRAINS N Subset for alpha = 0.05

1 2 3 4 5

A09108 3.00 1.54

A06115 3.00 1.64

A05113 3.00 2.22 2.22

A07301 3.00 2.33 2.33 2.33

A06112 3.00 3.18 3.18

Duncana A04127 3.00 3.32 3.32

A27302 3.00 3.37 3.37

A21108 3.00 3.50

A08201 3.00 4.88

A06116 3.00 6.67

Sig. .19 .07 .07 1.00 1.00

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

46